A quinone-dependent phosphatase was previosly purified to homogeneity from Clostridium sticklandii. The recent observations that 32P could be cleaved from both (32P) phosphocasein and (32P) phosphohistone suggest that the natural substrate of this enzyme is a bacterial phosphoprotein. After selective removal of the phosphatase from crude C. sticklandii extracts by passage over a quinone affinity column, the resulting preparations were labeled with 32P and surveyed as substrate for the phosphatase.